Journal of Experimental Botany, Vol. 47, No. 303, pp. 1577-1585, October 1996
Journal of
Experimental
Botany
Isolation and characterization of a new non-toxic
two-chain ribosome-inactivating protein from
fruits of elder (Sambucus nigra L.)
Lucia Citores1, Fernando M. de Benito1, Rosario Iglesias1, J. Miguel Ferreras1, Pilar Jimenez 1 ,
Pablo ArgUeso2, Gustavo Farias3, Enrique Mendez 3 and Tomas Girbes 14
1
Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias, E-47005 Valladolid, Spain
2
Instituto de Oftalmobiologia Aplicada, Universidad de Valladolid, E-47005 Valladolid, Spain
Servicio de Endocrinologi'a, Centro Ramdn y Cajal, Carretera de Colmenar Viejo km 9, E-28034 Madrid, Spain
3
Received 8 January 1996; Accepted 18 June 1996
Abstract
Introduction
Sambucus (Caprifoliaceae) species contain nigrin b
and ebulin I, which are two-chain ribosomeinactivating proteins (RIPs) belonging to a new type of
RIPs which are non-toxic to mice and cultured human
cells. In this work the presence in fruits of elder (S.
nigra L.) of a new non-toxic type 2 RIP (nigrin f) that
co-exists with a lectin known as SNA IV is described.
Nigrin f strongly inhibited protein synthesis in mammalian, but not in plant, ribosomes, promoting the depurination of sensitive ribosomes and thus allowing the
release of the RIP diagnostic RNA fragment. Nigrin f
is composed of two dissimilar subunits linked by
disulphide bridges with apparent Mr values of 31 600
and 26 300. The N-terminal amino acid sequence
revealed close homology of the catalytic A chain with
type 1 RIPs, especially those from Cucurbitaceae, and
the B chain with several lectins previously isolated
from Sambucus species. Nigrin f was not toxic to mice
when injected intraperitoneally up to 2 m g k g ~ 1 . In
addition, NHC human cells were also insensitive to
nigrin f up to 60 / / g m l ~ 1 . Anti-nigrin b rabbit polyclonal antibodies reacted with nigrin f, indicating that
nigrin b and nigrin f are proteins with similar
structures.
Some plants contain protein translational inhibitors collectively named ribosome-inactivating proteins (RIPs; for
a recent review see Barbieri et al., 1993). These proteins
display a N-glycosidase activity on large ribosomal RNA
of mammalian (Endo and Tsurugi, 1987), plant (Taylor
and Irvin, 1990; Prestle et al, 1992*; Iglesias et al, 1993*
; Bonness et al, 1994), yeast (Stirpe et al., 1988), and
bacterial (Hartley et al., 1991; Prestle et al., 1992a; Iglesias
et al., 1993a; Girbes et al., 1993a; Ferreras et al., 1994a,
b) sensitive ribosomes, thus promoting an irreversible
impairment of protein synthesis. The molecular action of
plant RIPs involves the hydrolysis of one (Barbieri et al.,
1993) or more (Iglesias et al., 19936 ; Barbieri et al.,
1992, 1994) adenines from the rRNA.
The classification of RIPs includes at least two categories: types 1 and 2 (Barbieri et al., 1993). Type 1 RIPs
consist of a single enzymic polypeptide chain. Type 2
RIPs contain two polypeptide chains linked by disulphide
bonds, one being the enzymic chain, which is equivalent
to a type 1 RIP, and the other a D-galactose and Dgalactosamine-binding lectin. Recently, a new RIP with
an unusual molecular structure and specificity for
Neu5Ac(a-2,6)-Gal/GalNAc has been described (van
Damme et al., 1996a). Four- and eight-chain RIPs are
special type 2 RIPs that are composed of either two or
four dimers, each one being composed of two polypeptide
chains linked by disulphide bonds (Barbieri et al., 1993;
van Damme et al ., 1996a). Type 2 RIPs, such as ricin
and viscumin, are able to associate to give tetramers
(Barbieri et al., 1993). Type 2 RIPs can also be divided
Key words: Sambucus nigra, elder fruits, nigrin f, ribosomeinactivating protein, characterization.
4
To whom correspondence should be addressed. Fax: +34 83 42 30 82.
O Oxford University Press 1996
1578
Citores et al.
into two categories: the toxic and non-toxic RIPs (Citores
et al, 1993). Type 2 RIPs are very infrequent and since
the isolation of ricin in 1888 by Stillmark, only a few
toxic type 2 RIPs (ricin, abrin, modeccin, viscumin,
volkensin, and Eranthis hyemalis lectin) have been isolated
(Barbieri et al, 1993; Kumar et al, 1993). Non-toxic
type 2 RIPs are even rarer and only ebulin 1 (Girbes
et al, 1993c) and nigrin b (Girbes et al, 1993ft) have
been isolated to date. Furthermore, the presence of a seed
isoform of nigrin has been reported in elder seeds (Citores
et al, 1994). Toxic type 2 four-chain RIPs are Ricinus
communis four-chain agglutinin and Abrus precatorius
four-chain agglutinin and non-toxic type 2 four-chain
RIP is the Viscum album four-chain agglutinin (Citores
et al, 1993).
Although the biological role of RIPs in plant producers
is presently unknown, based on the extracellular location
of the pokeweed antiviral protein from Phytolacca americana they have been proposed as antiviral agents (Taylor
and Irvin, 1990; Bonness et al, 1994; Ready et al, 1986;
Taylor et al, 1994). In this hypothesis homologous RIPs
would enter the virus-infected cell, thus triggering ribosome inactivation and therefore cell death (Bonness et al,
1994; Taylor et al, 1994). RIPs display antiviral action
on both animal and plant viruses containing either DNA
or RNA as their genome (Kumar et al, 1993; FoaTomasi et al, 1982; McGrath et al, 1989; Zarling et al,
1990; Lee-Huang et al, 1990, 1991a, ft; Lodge et al,
1993) in good agreement with Irvin's hypothesis. As
alternative, and probably working together, the direct
enzymic actions of some RIPs on TMV genome RNA
and herring sperm DNA have been recently described
(Barbieri et al, 1994; Girbes et al, 1996). Moreover, the
application of mediators of systemic acquired resistance
promote the expression of two type 1 RIPs named beetins
in sugar beet (Girbes et al, 1996), thus supporting the
hypothesis of antiviral function of RIPs.
Some RIPs share amino acid sequence homology with
topoisomerase II from Drosophila (Lee-Huang et al,
1994). This led to the finding that some RIPs display
topoisomerase activity that is exerted at rather high RIP
concentrations (Lee-Huang et al, 1994).
Due to their use as a toxic moiety in immunotoxins for
cancer (Olsnes et al, 1989; Brinkmann and Pastan, 1994)
and AIDS (Zarling et al, 1990; Till et al, 1989) treatment,
interest in RIPs is increasing rapidly. Moreover, it has
been shown that some RIPs have a direct inhibitory
action on HIV-1 replication (McGrath et al, 1989;
Zarling et al, 1990; Lee-Huang et al, 1990, 1991a, b).
The presence has recently been described of a novel
family of non-toxic type 2 (Girbes et al, 19936, c; Citores
et al, 1994) and type 1 RIPs (de Benito et al, 1995) in
Sambucus spp. It was observed then that the B chain of
these type 2 RIPs shares strong amino acid sequence
homology with some lectins isolated from Satnbucus spp.
(Girbes et al, 1993ft, c). In particular, it has been reported
that elder {Sambucus nigra L.) fruits contain a monomeric
lectin named SNA IV that is able to dimerize and
oligomerize (Mach et al, 1991).
Very recently, SNA I has been found to be also a type
2 RIP (van Damme et al, 1996a). Futhermore, SNA I
and nigrin b have been cloned and their structure elucidated (van Damme et al, 1996a, ft).
This work reports that S. nigra fruits contain a new
non-toxic type 2 RIP that has been named nigrin f. This
new RIP coexists with SNA IV (Mach et al, 1991) and
is both functionally and structurally related to the type 2
RIPs ebulin 1 (Girbes et al, 1993c), nigrin b (Girbes
et al, 1993ft) and SNA I (van Damme et al, 1996a).
Materials and methods
Materials
All chemicals and biochemicals were of the highest purity
available. Their sources are described elsewhere (Girbes et al,
1993ft, c; Citores et al, 1994). Sepharose 6B and Sephadex
G-25 were purchased from Pharmacia Iberica (Madrid, Spain).
DEAE-cellulose was obtained from Sigma (St Louis, MO,
USA). Acid-treated Sepharose 6B was prepared by treatment
of Sepharose 6B beads with 0.1 N HC1 at 50 °C for 3 h and
extensive washing with Milli-Q water (Girbes et al, 1993c).
Immobilon membranes were purchased from Millipore Iberica
(Madrid, Spain). Reagents for immunology were purchased
from Sigma (St Louis, MO, USA). L-[3H]valine (sp. act. 32
Cimmol" 1 ) was purchased from Amersham Iberica (Madrid,
Spain). [35S]Translabel (sp. act. 1164 Ci mmol" 1 ) was obtained
from 1CN through Nuclear Iberica (Madrid, Spain). Pokeweed
antiviral protein from seeds (PAP-S) was a generous gift from
Professor F Stirpe (Dipartimento di Patologia Sperimentale,
Universita di Bologna, Bologna, Italy). Green and mature fruits
of S. nigra were harvested from Cobos de Cerrato (Palencia,
Spain) in September.
Purification of nigrin f
100 g of either green (4 mm average diameter) or mature fruits
(5 mm average diameter) were cut into small pieces and then
ground in a blender and extracted overnight with 500 ml of
extraction buffer (280 mM NaCl containing 5 mM sodium
phosphate (pH 7.5). With this procedure elder seeds remained
intact, thus avoiding contamination of fruit extracts with seed
proteins. The extract was filtered through cheesecloth and the
fluid was then centrifuged at 25 900 x g for 45 min at 0 °C. The
resulting clear crude protein extract was filtered again through
cheesecloth to remove the fat layer that had developed. This
extract was subjected to affinity chromatography on acidtreated Sepharose 6B (AT-Sepharose 6B) pre-equilibrated with
extraction buffer at 4 °C. The protein was applied to the column
( 1 0 x 5 cm) and the unretained protein fraction was discarded.
The column was then washed with the same buffer until
absorbance reached the baseline value. The bound protein was
desorbed by elution with 0.2 M lactose in the extraction buffer
and dialysed against 5 mM sodium phosphate (pH 7.5).
Thereafter, the protein solution was applied to a DEAEcellulose column (5.6 x 2.6 cm) equilibrated with the same buffer
and fitted to an FPLC (Pharmacia) apparatus. After washing
with the same buffer, the column was eluted with the same
Nigrin f, an elderberry
buffer this time containing a gradient of 200 ml of 0-0.3 M
NaCl. This chromatographic step resolved two small protein
peaks at the start of the gradient and a major protein peak
spanning almost the whole gradient. Fractions containing the
protein peaks were pooled, dialysed against Milli Q-purified
water and finally freeze-dried.
Analysis of proteins by SDS-polyacrylamide gel electrophoresis
and detection ofglycan chains
Analyses of proteins by SDS-polyacrylamide gel electrophoresis
were carried out as described by Laemmli (1970) using 15%
acrylamide gels and the Mighty-Small II system from Hoefer
(San Francisco, CA, USA; technical bulletin No. 134). The
standards used were trypsin inhibitor (MT 20 100), carbonic
anhydrase (Mt 29 000), alcohol dehydrogenase (M r 37 000),
glutamate dehydrogenase (MT 54 000) and bovine serum
albumin (M r 68 000). The presence ofglycan chains was studied
using the Glycan Detection Kit from Boehringer (Mannheim,
Germany) using transferrin (Mr 79 500) as standard.
Protein synthesis
Polypeptide synthesis was carried out in cell free translation
systems derived from rabbit reticulocyte lysates, rat liver, wheat
germ, Vicia sativa germ, and Cucumis sativus germ following
current procedures (Girbes et al., 19936, c). The cell-free
extracts were filtered through Sephadex G-25 (8x2.6 cm) to
remove low A/r compounds and stored under liquid N 2 until
use and were thawed only once. In all cases translations were
carried out encoded by endogenous messengers since under
such conditions ribosomal sensitivity to RIPs has been shown
to be maximal (Arias et al., 1993).
Protein synthesis in NHC cells was carried out using
[33S]Translabel (sp. act. 1164 Ci mmol"') as described elsewhere
(Girbes et al., 1993c).
Analysis of 28 S rRNA N-glycosidase activity
100 /xl of rabbit reticulocyte lysate containing 20 mM TRIS-HC1
(pH 7.8), 50 mM KC1, 1 mM MgCl2, and 5 mM dithiothreitol
were incubated with 5 fig of nigrin f or known RIPs for 30 min
at 37 °C. Reactions were stopped by the addition of 500 /zl of
0.5% SDS containing 50 mM TRIS-HC1 (pH 7.6). RNA was
extracted by phenolization and ethanol precipitation. Aniline
treatment was carried out as follows: RNA was dissolved in 20
fA of water and incubated with 1 vol. of 2 M aniline (pH 4.5)
at 4°C in the darkness for 10 min. The reaction was stopped
by dilution with 400 /xl of water, and the aniline was removed
by two extractions with 1 vol. of ether. RNA was recovered by
ethanol-precipitation. Electrophoresis of RNA was carried out
in 5% acrylamide gels at 15 mA for 110 min as described
elsewhere (de Benito et al., 1995). The gels were photographed
after staining for 20 min with 0.5 ^g ml" 1 of ethidium bromide.
NH2-terminal amino acid sequence analysis
Nigrin f was subjected to SDS-PAGE in the presence of
2-mercaptoethanol to dissociate the A and B chains. Following
this, proteins were electroblotted on to PVDF membranes and
detected by staining with 0.2% Coomassie blue R-250 (w/v) in
methanol:water:acetic acid (50:40:10, by vol.), for 0.5-2 min.
The membranes were then washed in methanol: water: acetic
acid (48:47:5, by vol.), and the well-defined protein bands
were cut out of the PVDF and applied to a KNAUER sequencer
with an on-line PTH amino acid analyser according to
manufacturer's instructions.
RIP
1579
Enzyme-bnked Immunosorbent Assay for nigrin f and related
proteins
ELISA was performed essentially using the method outlined in
Perez-Maceda et al. (1991). Detection was performed with antinigrin b rabbit polyclonal antibodies (67 pg per well) as the
first antibody, obtained as described elsewhere (Harlow and
Lane, 1988) and partially purified by chromatography through
protein-A Sepharose 4B (Pharmacia-LKB), as described
(Harlow and Lane, 1988). Alkaline phosphatase-linked goat
anti-rabbit antibodies (Sigma, St Louis, MO, USA) were used
as the second antibody. Substrate treatment and colour
development were carried out as indicated elsewhere (PerezMaceda et al., 1991). Absorbance was measured at 405 run in
an ELISA reader (LP400 from Pasteur Diagnostics).
Measurements were made in quadruplicate.
Red blood cells agglutination
Red blood cell agglutination was carried out at room temperature in microtitre plates containing 100 fA of 5 mM sodium
phosphate (pH 7.5), 0.14 M NaCl and 0.5% of red blood cells.
Results
Isolation of nigrin f and SNA III from green and mature fruits
ofS. nigra
The rationale of our search in elder fruits came from
preliminary experiments that indicated the presence of
translational inhibitory activity in crude extracts of green
elder fruits (Table 1). From our own data (Girbes et al.,
1993ft, c), it was concluded that these could contain a
new type 2 RIP. To isolate this possible inhibitor, crude
protein extracts were chromatographed by an affinity
process as reported previously (Girbes et al., 1993c). As
shown in Fig. la, elder fruit protein extracts contained Dgalactose-binding proteins that bind to acid-treated
Sepharose 6B and can be detached by 0.2 M lactose, in
agreement with the results of Mach et al. (1991). These
protein extracts strongly inhibited protein synthesis
(Table 1). Attempts to resolve the D-galactose and Nacetylgalactosamine-binding proteins by exclusion chromatography were unsuccessful due to the presence of a
major single-chain protein revealed as the previously
reported lectin SNA IV (Mach et al., 1991). A similar
problem has been found for the isolation of ebulin 1 from
leaves of 5. ebulus. In this case, the RIP was retained by
the Superdex 75 matrix, thus eluting as a protein with an
apparent Mr of around 30000. With nigrin f and SNA
IV this prevents protein resolution by size exclusion
chromatography. The strategy was therefore changed and
ion exchange chromatography tried. As shown in Fig.lb,
a salt gradient (0-0.3 M NaCl in buffer phosphate) in
DEAE-cellulose afforded the resolution of two small
protein peaks at the start of the gradient and a major
protein peak that spanned almost the whole gradient. As
reported below, the first two peaks (nigrin f) strongly
1580
C/fores et al.
Table 1. Purification of nigrin f from green and mature Sambucus nigra fruits
The protein was purified as described in Materials and methods. Inhibition of protein synthesis was performed using a rabbit reticulocyte cell-free
system. One unit (U) is the amount of inhibitory protein that reduces 1 ml translation reaction mixture by 50%
Protein
(mg)
Step
Green fruits
Crude
AT-Sepharose
DEAE-Cellulose
Mature fruits
Crude
AT-Sepharose
DEAE-Cellulose
(ngml" 1 )
Specific activity
(x 10" 3 Umg" 1 )
Total activity
(x 10" 6 U)
Yield
2640
90
1.3
58
26
1.8
17.2
38.5
556
45.5
3.4
0.7
100
7.5
1.5
2930
13
0.12
190
145
1.9
5.3
6.9
526
15.4
0.9
006
100
5.8
0.4
200
400
ELUATE (ml)
100
ELUATE (ml)
150
200
0.0
Fig. 1. Isolation of nigrin f from green fruits of Sambucus nigra L. Nigrin f was isolated as described in Materials and methods, (a) Affinity
chromatography of crude protein extract from green fruits of 5. nigra. The arrow indicates addition of 0.2 M lactose, (b) DEAE-cellulose-FPLC
of protein from the affinity chromatography step Horizontal bars indicate fractions that were pooled and subjected to further studies.
inhibited protein synthesis in a rabbit reticulocyte lysate
while the third peak (SNA IV) proved to be largely
inactive as a translation inhibitor.
Molecular structure analysis
As shown in Fig. 2, SDS-PAGE analyses of these peaks
in the absence of 2-mercaptoethanol (2-ME) revealed
that the first protein peak contains a homogeneous protein
that migrates with an apparent MT of 58 000. This was
named nigrin f and, and as shown in this paper, it is a
new type 2 RIP. The second protein peak contained the
same protein although it was contaminated with a small
amount of the protein from peak three. Peak three
contained a homogeneous protein that migrates at an
apparent Mr of 30000 and that, on the grounds of
previously published properties and our own results, is
the SNA IV lectin reported earlier (Mach et al., 1991).
SDS—PAGE analyses of the protein peaks in the presence of 2-ME (to break disulphide bridges) revealed that
nigrin f has four proteins: two major ones with apparent
Mr values of 31 600 and 26200, and another two minor
ones with apparent Mt values of 30000 and 28 200. SNA
IV is a polypeptide chain with apparent MT of 30000
accompanied by a very minor and closely related band
exhibiting faster migration properties. Isoelectrofocusing
analysis revealed that nigrin f has two significant isoforms
one of them major and other one almost negligible (data
not shown). These data are consistent with a major
structure with a MT of 58000 (MT 26300 plus Mr 31 600)
and a minor structure with a Mr of 58 000 (MT 28 000
plus Mr 30000). By analogy with ebulin 1 and nigrin b,
and as confirmed by N-terminal amino acid sequence
analysis (see below), the protein of MT 26 300 would be a
catalytic A chain while the protein of MT 31 600 would
be a lectin moiety able to bind to D-galactose residues.
As shown in Fig. 2b, the B chains of nigrin b -the type
2 RIP from elder bark- and nigrin f are both highly
glycosylated proteins, as revealed by the glycan detection
assay. Additionally, SNA IV is also strongly glycosylated,
in agreement with Mach et al. (1991).
Nigrin f, an elderberry RIP
A
2ME
+
-
Mr
1 2
B
+
+
3
1
2
1581
3
4 1 3 Mr
100 >
tt
III
I
68.
543729-
- »••40
c
o
o
• Mm
UJ
Fig. 2. Analysis of D-galactose-binding proteins from 5. mgra fruits by
SDS-PAGE. Panel A: Coomassie blue staining. Protein fractions (13
Hg per well) were electrophoresed in the presence or absence of
2-mercaptoethanol (2ME) and then stained with Coomassie blue for
visual assessment, as indicated in Materials and methods. The numbers
on the left indicate the corresponding molecular masses of the standards
in kilodaltons. Panel B: Glycan detection. Protein fractions (3 fig per
well) were electrophoresed in the presence of 2-mercaptoethanol,
transferred to an Immobilon P membrane and detected as indicated in
Materials and methods. Lane numbers (1 and 3) indicate the
corresponding peaks in Fig. 1 and lane 4 indicates nignn b. The number
on the right indicates the corresponding molecular mass of the standard
in kilodaltons.
Effect of elder fruit maturation on protein content
Mature fruits of elder displayed a similar pattern of
proteins as green fruits but with much less protein. The
steps and yields of the isolation procedure of nigrin f and
SNA IV from mature fruits are also outlined in Table 1.
The total content of inhibitory activity, much of which is
lost during the isolation of nigrin f, is striking. This is
probably due to the presence of inhibitory compounds
such as RNases, polyphenols, pigments, etc., and of type
1 RIPs named nigritins (manuscript in preparation) which
are similar to those described recently in the leaves of
dwarf elder (Sambucus ebulus L.) (de Benito et al., 1995).
Additionally, there is a strong reduction in the amounts
of nigrin f and SNA IV contents (10-fold) in mature
fruits with respect to the green fruits.
Inhibition of protein synthesis
To define nigrin f as an RIP, its effects on translation in
vitro were analysed. As shown in Fig. 3, nigrin f strongly
inhibited protein synthesis carried out by rabbit reticulocyte lysates and rat liver cell-free systems while it had no
effect on plant cell-free systems derived from wheat, V.
sativa and C. sativus germs. Its inhibitory potency is well
defined by its /C 5 0 (concentration of protein that promotes
a 50% inhibition of protein synthesis). Nigrin f/C5Os were
1.8 and 3.7 ng ml" 1 for rabbit reticulocyte lysates and rat
liver cell-free systems, respectively. These values are lower
than those reported for ebulin 1 (Girbes et al., 1993c) and
nigrin b (Girbes et al., 19936), such differences being
highly reproducible among the different batches of nigrin
f. The effects of nigrin f on protein synthesis carried out
by NHC cells of human origin were also analysed. The
>U1
o
a:
a_
25-
o1-
10°
102
1
[TOXIN] (ng/ml)
Fig. 3. Effects of nignn f on protein synthesis carried out by several
eukaryotic cell-free translation systems. Protein synthesis inhibition by
nigrin f was assayed in rabbit ( • ) , rat (O), Vicia sativa ( • ) , Triticum
aestivum ( • ) and Cucumis salivus (A) cell-free translation systems, as
indicated in Materials and methods. Controls were run in the absence
of inhibitor.
results (data not shown) indicated that up to a concentration of 60 /xg ml" 1 , nigrin f was inactive on such cells in
contrast to the highly toxic type 2 RIP ricin (Olsnes and
Pihl, 1982). Additionally, and to confirm the data on
cultured cells, up to 1.56 mg kg" 1 of body weight of
nigrin f to mice was injected with no apparent toxic
effects over 20 d. However, derangement of non-vital
processes cannot be excluded in such de visu analyses.
N-glycosidase activity
In order to confirm the inhibitory activity of nigrin f on
mammalian ribosomes, the mechanism of action of the
protein was investigated. Accordingly, the N-glycosidase
activity characteristic of RIP action (Endo and Tsurugi,
1987) was analysed on rabbit reticulocyte ribosomes. As
shown in Fig. 4, treatment of rabbit reticulocyte lysates
with nigrin f for 30 min, which as shown in Fig. 3
inactivates ribosomes, promoted the depurination of ribosomes, which upon rRNA isolation and subsequent treatment with acid aniline splits the RNA fragment diagnostic
of RIP action (Endo and Tsurugi, 1987). Such inactivation occurs through depurination of the 28 S rRNA at
the highly conserved adenine responsible for the interaction of elongation factors with the ribosome, in both
eukaryotic (Brigotti et al., 1989) and prokaryotic organisms (Iglesias et al., 19936). The same result was obtained
with pokeweed antiviral protein (PAP-S), a type 1 RIP
1582
Citores et al.
Control Nignn f
Aniline
+
PAP-S
-
Typt 1 mp» and tvp. 2 BIP-A ch«ln«
+
lII
Ogrln f A-chaln
Higi-In b A-chaln
Ebulin I A-chain
Fig. 4. rRNA jV-glycosidase activity of nigrin f rRNA jV-glycosidase
activity was assayed as indicated in Materials and methods. Each lane
contains 3 /ig of RNA. The arrow indicates the RNA fragment released
as a consequence of RIP action upon acid aniline treatment.
obtained from seeds of Phytolacca americana (Barbieri
et al., 1982).
Agglutination of human red blood cells
Type 2 mP-» dialnt and Lactlrn
Hlgrln f B-chaln
algrln b B-chaln
Both nigrin f and SNA IV promote the agglutination of
human red blood cells at quite low concentrations. Total
agglutination by nigrin f of red cells from type O blood
was achieved at 58 ^g ml"' and from type AB blood at
116 /xg ml" 1 . SNA IV isolated by this procedure agglutinated red cells from type O blood at 88 ^gml" 1 . These
values lie within the range of those reported for SNA II
(Kaku et al., 1990) and the lectin described in elder seeds,
also named SNA III by Peumans et al. (1991). Both of
these are iV-acetylgalactosamine-specific lectins and their
agglutination capacities are much lower than those
reported for SNA I which is a sialic acid-specific lectin
(Broekaert et al., 1984).
N-terminal amino acid sequence of nigrin f
In order to compare nigrin f with known proteins, in
particular the SNA IV from elder fruits (Mach et al.,
1991), and the RIP SNA I from elder bark (van Damme
et al., 1996a), the N-terminal end of nigrin f was
sequenced. As shown in Fig. 5, the nigrin f-A chain shares
strong amino-acid sequence homology with the A chain
of both ebulin 1 (Girbes et al., 1993c), nigrin b (Girbes
et al., 1993/)) and SNA I (van Damme et al., \996a), and
also with several type 1 RIPs such as trichosanthin
(Collins et al., 1990), TAP 29 (Lee-Huang et al., 1991a),
bryodin-S (Montecucchi et al., 1989), luffin a (Funatsu
et al., 1988), all from Cucurbitaceae, and gelonin
(Montecucchi et al., 1989) and ricin-A chain (Lamb et al.,
1985), from Euphorbiaceae and PAP (Kung et al., 1990)
and an internal region of topoisomerase II from
Drosophila (Lee-Huang et al., 1994). The nigrin f B-chain
also shares strong amino acid sequence homology with
the B-chains of nigrin b (Girbes et al., 19936), ebulin 1
(Girbes et al., 1993c), SNA I (van Damme et al., 1996a)
and ricin (Araki and Funatsu, 1987) and also with the
Ebulin I B-chaln
Klein D B-chaln
SKA I I I
HA li
SKA I B-chaln
(
Fig. 5. N-terminal amino-acid sequences of nigrin f. jV-terminal amino
acid sequence analysis was carried out as described in Materials and
methods. The boxes enclose identical amino-acids. Protein sequences
were taken from the references indicated in text.
lectins SNA IV (Mach et al., 1991) and SNA II (Kaku
et al., 1990). These results clearly indicated that nigrin f
isolated here is a protein other than SNA IV.
Immunological cross-reactivity of nigrin f with anti-nigrin b
rabbit polyclonal antibodies
As shown in Fig. 6, ELISA analysis of non-toxic type 2
RIPs revealed that anti-nigrin b rabbit polyclonal antibodies at a dilution of 1/3500 strongly reacted with nigrin
f. By contrast, these antibodies showed only a weak
reaction with several lectins isolated from S. nigra such
as SNA II (Kaku et al., 1990) and IV (Mach et al., 1991)
and the RIP SNA I (van Damme et al., 1996a).
Discussion
A recent paper dealing with the finding of a new lectin
reported that elder fruits contain a new lectin, named
SNA IV, that tends to form di- and oligomeric aggregates
(Mach et al., 1991). Here, the presence of SNA IV in
elder fruits has been confirmed, but have found among
the dimeric aggregates of SNA IV a novel non-toxic type
2 RIP of the ebulin 1 (Girbes et al., 1993c) and nigrin b
Nigrin f, an elderberry RIP
20
E
C
in
o
15
U
O
m
<x
o
(/>
10
CD
05
5
10
15
20
[PROTEIN] (ng)
25
Fig. 6. Immunological cross-reactivity of rugrin f with anti-nigrin b
rabbit polyclonal antibodies. Immunological cross-reaction with antinigrin b was studied by ELISA, as described in Materials and methods.
Symbols: ( • ) nigrin b; (O) nigrin f; (A) SNA I; (A) SNA II; ( • )
SNA IV. The points represent the means from four sets of measurements
and bars indicate the standard error of the mean.
(Girbes et al., 19936) family. This compound has been
named nigrin f.
Nigrin f contains two different polypeptide chains that,
in view of all the data on protein synthesis, electrophoretic
analysis, amino acid sequence and red blood cell agglutinating activity, are clearly the catalytic A chain (M r 26200)
and the lectin B chain (A/r 31 600), characteristic of
common type 2 RIPs.
Nigrin f fulfils all requirements to be classified as a
non-toxic type 2 RIP that strongly resembles, but is
clearly different from the other two non-toxic type 2 RIPs
reported to date: nigrin b (Girbes et al., 19936) and
ebulin 1 (Girbes et al., 1993c). The small changes in the
amino acid sequence of nigrin f with respect to nigrin b
and ebulin 1 indicate that all of them derive from a
common ancestor, also probably related to the lectins
SNA II (Kaku et al., 1990) and SNA IV (Mach et al.,
1991). It is very interesting to note the close correlation
between the nigrin f A-chain and type 1 RIPs of
Cucurbitaceae (a genus very close to the Caprifoliaceae)
and the ricin A-chain (Euphorbiaceae), which display
potent anti-HIV-1 effects (McGrath et al., 1989; LeeHuang et al., 1990, 1991a, b\ Olson et al., 1991). However,
to the best of our knowledge no studies have reported
the presence of equivalent non-toxic type 2 RIPs or
structurally related lectins in these genera.
Nothing is known of the biological role of nigrin f,
which is the case of RIPs in general. Nonetheless, it has
1583
been suggested that SNA I, a sialic acid and galactosebinding lectin with RIP activity from elder bark probably
related to the structure of nigrin f B-chain, as found with
other lectins isolated from elder (Mach et al., 1991; Kaku
et al., 1990), is located in the protein bodies of phloem
parenchyma of bark (Nsimba-Lubaki and Peumans,
1986). On these grounds, the involvement of SNA I in
protein storage processes was suggested. These results
indicate that maturation of dwarf elder fruits leads to a
strong reduction in the content of both nigrin f and SNA
IV (Table 1). This is also consistent with a storage role
for both proteins. However, it remains unclear whether
nigrin f might trigger plant cell maturation or whether
such a reduction would merely be a consequence of
maturation. On the other hand, these results also indicate
that anti-nigrin b rabbit polyclonal antibodies react in
Western blot analysis with SNA I and SNA II (data not
shown), thus pointing to a close structural correlation
between the elder RIP and the elder lectins. Further work
will address the potential antiviral properties of nigrin f
on the basis of its primary structure and activity on RNA.
Acknowledgements
This work was financially supported by research grants from
the Comision Interministerial de Ciencia y Tecnologia (CICYT)
-Grants BIO92-0231, PTR94-0062 and BIO95-0610- and
Consejeria de Sanidad y Bienestar Social (Convenio 1995, Junta
de Castilla y Le6n). L Citores holds a predoctoral fellowship
from Ministerio de Education y Ciencia. We wish to thank Mr
N Skinner for correcting the English version of the manuscript
and JE Basterrechea for his technical support.
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